JPH04506562A - How to prevent papilloma virus (HPV) in the human body for medical purposes - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] A method for preventing papilloma virus (HPV) in the human body for medical purposes.

This invention relates to a method for preventing papillomavirus (HPV) infection in the human body for medical purposes. It is something to do.

Cervical papillomatous virus infection is associated with cervical carcinoma and is more than O50 type. HPV that can be used has already been identified. HPV types 6, 11, 16, 18, 31, 33 and 51 have been found to infect the reproductive tract. Types 6 and 11 are Type 16, which causes gradual growth foci on genital organs and condyloma-like acupoints. 18, 31 and 33 are found in pre-cancerous lesions and often in cervical cancer types. . Antiserum 6 that reacts with bovine papillomas and viruses of any group Immunology The related human nipple swelling can be easily prepared. Against virus protein protective membrane Papilloma protein protection by using group-specific antisera generated as Membrane antigens are found in precancerous systems and fungal muscles. genital warts, genital warts Patients with genital epithelial neoplasia (CIN) and genital tumors are group-specific protein carriers. had higher serum IgG antibody levels than the comparison group for protective membrane antibodies. It has been reported.

It is an object of the present invention to provide a method as described above for the treatment of carcinomas, particularly cervical or precarcinomas. The goal is to enable early detection of cancer symptoms and avoid the risk of culturing cancer.

Another object of the invention is to detect the presence of antibodies in body fluids against HPV. Easily and quickly identify the risk of carcinoma or precancerous symptoms or carcinoma culture. The purpose is to provide a method.

Therefore, in this invention, as described in claims 1.11.21 and 31, It has unique characteristics.

The present invention will be explained in more detail below with reference to the drawings.

Figure 1 is a graph showing the detection of IgA antibodies against papilloma viruses in cervical secretions. centre, Figure 2 shows the correlation of IgA antibodies against papilloma viruses in serum and cervical secretions. A graph showing the relationship, Figure 3, shows the relationship between papilloma viruses in serum and genital secretions. Figure 4 shows the comparison of IgG antibodies in genital secretions. Figure 5.6 is a graph showing a comparison of IgG and IgA antibodies against Detection of IgG and IgA antibodies by blotting Figure 7 shows the age of serum IgA antibodies against pv in the normal population. Graph showing gender distribution, Figure 8 shows the age-sex distribution of serum IgG antibodies against Pv in the normal population. graph, Figure 9 shows Ig against Pv in serum from patients with CIN or cervical carcinoma. Graph showing the detection of A antibody, Figure 10 shows whether the patient had CIN or cervical carcinoma. Figure 11 is a graph showing the detection of IgG antibodies against PV in the serum of these patients. or detection of IgM antibodies against Pv in serum from patients with cervical carcinoma. Figure 12 shows the reaction between IgA and IgG to Pv in relation to disease. Graph showing the difference, Figure 13 is a graph showing IgA reactivity to L1 protein; Figure 14 is a graph showing IgA reactivity to L2 protein; Figure 15 is a graph showing IgA reactivity to L1 protein; Figure 16 is a graph showing IgG reactivity to L2 protein; Figure 17 shows 1gM reactivity for Ll protein. Figure 18 shows L2 protein reactivity. Figure 19 shows the reaction of synthetic peptides with disease at 1 gM. Graph showing gender, Table 1A is a comparison of IgAS and IgG antibodies against pv in serum and cervical secretions. Table 1 is a graph showing detection of antibodies associated with cervical carcinoma, Table 2 is a graph showing detection of antibodies associated with cervical carcinoma. A graph explaining the formulas used for synthetic peptides, Table 3 shows the amino acids of synthetic peptides. Graph showing acid permutation, presence or absence of IgG antibodies against papilloma and progression of deterioration To investigate whether there is a relationship between Vagina secretions were examined by a modified EL ISA method.

Forty-two women between the ages of 20 and 50 participated in the study. They have abnormal vagina stains before had a tinnitus or fungi lome or participated in a screening program. all All patients underwent colscope examination. full bosco A rectangular contamination was taken on the day of the operation, and in some cases confirmed by colboscopy. A biopsy was taken from the lesion. cytological and histomedical Various tests were conducted and finally the results were compared with the ELISA results. Morphology of HPV infection The judgment was made by Meisel et al. (1979). Positive papilloma virus infection was suggested by koilocytotic cells. this Typical koilocytotic cells have an enlarged, hyperchromatic nucleus with a clear pattern. Surrounded by topplasmic regions. Vagina cervical lesion is endoect ( (endo-ecto) was collected from the neck with a small sterile swab. This disinfectant cotton The mixture was placed in a glass tube containing 0.5 ml of PBS and mixed using a vortex mixer. Ta. Samples were stored at 20°C until use. By weighing the tube before and after sampling. The exact amount of secretion was estimated. Before use, these were centrifuged to remove debris. . Samples were collected on any day during the menstrual cycle except on the day of actual menstruation. stained with blood The stained samples were discarded.

BPV was purified as follows. Glycerol phosph for raw wood skin lumps Store at +4°C in ultra-Tarrax mixer in ate buffered saline (1:1). - Homogenized in 0.1 M Tris-HCl. Uses 10% suspension After adding Taleosil and Freon, I added Susbenji Seal to Tsuruvar 5S-34. Centrifugation was performed at 11.950 g for 30 minutes using a 0-tar. More of what you got MSES Cutitan rotor medium 5. Centrifugation was performed at OOOr pm for 50 minutes. child The pellet of O, IM) was suspended in ICI and mixed with CsC1. equilibrium For 35. The solution was centrifuged overnight in a MSEI:liter at OOOrpm. . Collect the virus band and add it to TE buffer (0, OIM), Lis HCI, O, OO. OIMEDTA) and stored frozen at -70°C. skin lumps The extract was purified by two rounds of CaCl equilibrium centrifugation. two different bands Found in ingredients. The lighter band has a buoyant density of 1.2917’ml; When confirmed with an electron microscope (EM), it contained empty virus particles.

The heavy crawling band has a buoyant density of 1.34 g/ml and is typical of a papilla when examined by EM. It contained complete virus particles with tumor virus morphology.

Approximately 100 μg of purified virus suspended in Floyd's Complete Supplement Immunization of raw materials purified by intramuscular inoculation against papillomaviruses Serum was prepared for the rabbit. After 2 weeks using the same virus and without supplementary drugs. Two subsequent vaccinations were given. Rabbits were bled one week after the last vaccination.

A modification of the ELISA method described above was used. Dilute BPV virus in PBS Add to 96-well trace plate at a constant concentration of 0.06 μG/well. It was kept overnight at +4°C. After washing three times with PBS containing 0.05% Tween , plate with 4% Virus Dental Serum Albium (Eba) and O, 1% gelatin. Blocked for 6 hours at room temperature. Thereafter, it was left to stand at +4°C. plate before use Washed 5 times with PES Tween. Vagina secretion was diluted to 1/40, serum was diluted to 4 % BSA, 1/100 diluted in 0.1% Tween. To detect antibodies, Conveniently purified biotinylated anti-human IgA and IgG at 1/1000 at room temperature for 4 hours. It was diluted and used.

Peroxide avidin was added at a 1/400 dilution for 2 hours at room temperature. As a fabric 0.4% Aufetani! diamine, 0°02% H,O□ in 50mM phosphate. Esitrate buffer, pH 50, was used. Rabbit antiserum for positive comparison Using anti-BPV virus and/or rabbit antiserum against elk papilloma virus . New human serum and rabbit preimmune serum were used as negative comparisons. Ta.

BPV pillion (VIIIION) on a 5-20% polyacrylamide component gel. I made an electronic foray. Approximately 5 μg of pillion was applied to each sample. Tobin and others The sample was transferred to a nitrocellulose sheet by the method. Cut this sheet into strips and make 2% toy. 50mM at tone 80) Lis PH10, 2, 150mM NaCl, 5mM N It was left in aNs for 15 minutes. The nitrocellulose band was soaked overnight in the patient's buffer solution. /Zoo diluted serum.

- After one night the plots were washed in the same buffer. Rabbit antiserum against Pv Ditib was used as a comparison and rabbit preimmune serum was used as a negative comparison.

To detect bound antibodies, goat anti-human antibody or IgG is added to the buffer at 1/1000 concentration. It was used diluted. Next, the plot is peroxide avidin diluted 1740 left in G buffer. For rabbit antiserum, goat anti-rabbit 1gG peroxidide The sample was diluted 1/100o in buffer and used. All plots are 50mM sodi Incubated with carbazole in um acetate F'l (50).

Figure 1 shows detection of IgA antibodies against papillomavirus. Cervical secretions 1/4 0 was applied to an ELISA play coached with a purified pillion. IgA antibodies are Detected by IgA specific antibody and avidin peroxidase conduit gate did. Absorption at 490 nm was recorded 15 minutes after adding the dough. background Absorption values >0.1 were recorded as positive.

IgA antibodies against Pv were found in the cervical secretions of women aged 17-42. 1st In the graph shown, each point represents the average uptake of a sample from a patient. had ClN5 Nine women had IgA antibodies in cervical secretions. Koishito contaminated with Pap Tics and fluboscopically confirmed non-CrN symptoms were present in 9 patients. These three people had IZA antibodies. Six of the 24 women had normal tests. The results showed that the patient had IgA antibodies. IgAELISA absorption is The loops were examined for significant differences. 3 types of non-parametric tests The CIN group showed significantly higher absorption values than the normal group. . The proportion of IgA positive cervical secretions was also lower in the CIN group than in the normal group. It was observed that this was significantly higher. This comparison shows that the syndrome/phylocytosis There was no difference between the group with tics and the normal group ( p>0.05; absorption mean = 0.109 vs. 0.104).

We compared IgA and IgG levels in serum and secretions for 15 patients. . As shown in Figure 2, IgA antibody levels were relatively correlated. Figure 3 As shown in Figure 2, IgG antibody levels were not significantly correlated. As shown in Figure 4 No correlation was found between the two levels.

To determine which polypeptides the Pv antibodies are directed against, purified BPV Lyon immunoplotting was performed.

In Figure 5.6, purified BPV pillions were electrolyzed on 5-20% polyacrylamide. The child was foreseed and transferred to nitrocellulose. The obi is purified bobbin pillion, Human neoplasms and rabbit hyperimmune sera against 10 patient sera were included.

Goat anti-human IgG (Figure 5) or goat anti-human 1gA (Figure 6) and peroxidase Bound antibodies were detected by Cyd-Zavizi. In the rgA test, serum No. , the bands of patients 2-9 were negative as in N001. In Figure 5.6 Arrows indicate the main detected Pv proteins, K=Juyuki Kg1MW=marker It is molecular weight. The molecular weights of the markers are 200; 116; 92; 66; 44 and 2 It was 9 kilodaltons.

Rabbit hyperimmune serum prepared for purified bobbin pillion contains 4 types of polypeptides. (14 kDa protein, 28 kDa protein, 64 protein and 54 kDa protein) a protein) was detected. For the 54kDa protein, 10 people were tested. Ten of the patients had serum IgG antibodies. Two people had serum IgG antibodies of 64 kDa protein.

Two had serum IgG antibodies against the 28 kDa protein. IgA% constant According to the results of testing about Njinigate, only 1 out of 10 people could detect Ig. A antibody level was shown by immunoplotting (Fig. 6). By EL I SA However, this serum showed exceptionally high IgA anti-BPV reactivity.

In immunoplotting this is opposite to 14kDa, 28kDa and 54kDa. However, it reacted weakly with 64kDa.

The presence of localized genital papilloma virus antibodies that can be easily detected and measured. It was revealed that. IgA antibodies against Pv in cervical secretions and tissue forehead of CIN It became clear that there was a strong correlation between the diagnosis of However, 6 out of 24 He had a normal human Pap infection and a fluvoscopic body. have IgA antibodies However, patients who do not have CIN may have histologically undetectable CIN lesions. It is unknown whether or not it will occur. According to previous research, the papillary virus group has been identified. This suggests that IgG antibodies against the antigen are elevated in CIN. However, Pv Serum IgG antibody levels are also elevated in patients with skin bumps.

This issue can be addressed by measuring IgA described here and determining whether it is related to genital contractile Pv infection. This can be solved by measuring the antibodies. The tests described here are based on the I I would like to emphasize that there is no correlation with tests using gG. This means that in serum There is a correlation between IgA antibodies against HPV and secretions (Figure 2), There is a simple correlation between IgA and IgG antibodies in secretions or serum against PV. It is concluded from the fact that there is no such thing (Figure 4, Table 1). Level and content of IgA antibodies in serum There was a correlation between secretions. Therefore, IgA antibodies in serum cause genital contraction. There is a possibility that specific tests can be given that can be applied in daily medical care.

Normal subjects and patients with viral copies have IgA antibody levels similar to normal subjects. It's amazing that we have this. However, this situation What can also be seen about Steinbar (Epstein-Barr) Weels Here, the anti-virus protein protective membrane IgA antibody reaches the virus production level. is not relevant, only cancer with viruses. Immunoblotting is 4 A polypeptide with several pilions was detected. IgG antibody against 54kDa The existence of is consistent with previous research. 56kDa1gG immune protein code 11 orbs The leading frame was identified as the main component of the protein protective film. habo7 0kDa protein protective membrane protein is available in Food L2 open reading frame. be. This protein corresponds to the 64kDa protein we detected.

The identity of the 28kDa protein and the 14kDa protein is unknown. IgA alone Positip serum has an IgA reaction against 54.2g and a 14kDa polypeptide. However, the IgG reaction of this serum was directed only against the 54 kDa polypeptide. It was. From this, the Pv selection for high IgA and IgG is not always the same. I understand that.

To analyze serum antibody responses to group-specific PV protein protective membrane antigens, The proportion of serum antibodies to normal and healthy patients pv with CIN or cervical carcinoma compared with that of a woman who

A total of 139 comparison sera were obtained from healthy sexual women. I got a 62 from a woman in obstetrics and gynecology. . All women had no pathological findings or condylomata, but CI There was no histological evidence of N. A healthy woman who underwent regular annual serum testing of 59. Obtained from laboratory employees.

A group of 114 women with untreated histologically confirmed cervical tumors. 13 lesions were classified as CINI and 16 lesions were classified as ClN2. 16 lesions were classified as ClN3 and the remaining 69 lesions were classified as invasive cervical lesions. It was classified as a carcinoma. 83 sera from children of various ages and adults of both sexes were collected. Obtained.

Virus isolation and purification was performed as described above.

The preparation of the bobbin papilloma virus is shown in Figure 5.6. All four proteins are immune Human serum contained immunoreactive shrimp as shown in the plot.

The purified BPV was split by 5 times of cryo-sawing (thoa blazing). , diluted with 10mM carbonate buffer PH9.6, half Asia 96-wafer lumimicrotiter plate at a constant concentration of 0.15 μg/well. BPV The plates were kept at room temperature overnight and then washed with 0.05 PES) Ly-n20. The plates were then placed in 10% lamb serum in PES and left at 37°C for 60 minutes.

This solution was discarded and the plate was completely wrapped in paper. Human serum 10% lamb serum/ It was diluted 1:20 with PES, added to the plate, and reacted at 37°C for 120 minutes. So The plates were washed five times with PBS-T.

To detect bound antibodies, plate 120 minutes at 37°C. Horseradish bar oxidase monoco for human body 1gA diluted in serum/PBS A null antibody was used.

Next, wash the plates 5 times with PBS-T, and add 1 wash of 0.1M citrate acid. Nil: 20mg/m diluted by 50% The cells were cultured with diammonium salt (Jin-Sulfonat (6)). absorption is 30 It was 415 nm after minutes (Figure 7.8) or 60 minutes (Figures 9-12). I For detection of gG, plates were washed with 10 rum serum PBS for 60 min 37 It was left at ℃.

Then 10% lamb serum PES or monoclonal antibody against human 1gG hose Radish 1 peroxidase condi 1 in 1 gate: 12 at 30℃ to 1000 One gate of rabbit anti-human antibody 1gG alkaline phosphate diluted 0 min was used. (Figures 9-12). 0.1M Jetanolamine PH9゜6/1mM MgC Wash with PBS-T 1 mg/ml phosphatase fabric in l buffer, then pre-prepare. The samples were read at 405 nm after 9 g minutes. A baroxidase antibody was used. At times, plates were incubated for 30 minutes for IgA conjugation. IgM anti For body detection, the plate was left as described above and treated with 1 gM oxidase of anti-human body. with a 1:800 dilution in 10% lamb serum/PBS for 120 minutes. 6 After 0 minutes the plates were read at 415 nm. 0.1 for all EL I SAs Absorption of 5 or more was considered a positive response.

Two CIN patient sera with known reactivity were used as internal standards for all tests. there was. Multiple uncoated wells were used as negative comparisons.

Fluid absorption for stochastically significant differences between patient groups and healthy comparison groups The average was analyzed.

The results were as follows.

Out of 83 sera from children and male and female adults, 24 had IgA antibodies and 46 had IgG antibodies. had antibodies. IgA anti-BPV titars for healthy girls In comparison, it was observed that the ratio increased for healthy boys (Figure 7). This picture is normal Figure 2 shows the age-sex distribution of serum IgA antibodies among subjects. Serum was diluted 1:20 and IgA antibodies were detected for alpha chain specific peroxidase antibodies. It was done. Following the addition of the dough, absorption at 415 nm was recorded after 30 minutes. each The dots indicate the average absorbance of the samples with average comparisons for each patient. No gender In both cases, IgA antibody levels were very similar in children compared to adults.

The same sera tested for IgA antibodies showed no significant increase in IgG as shown in Figure 8. was also tested. This is the age- and sex-specific serum IgG content in healthy subjects. Showing cloth. The same serum was tested for the presence of IgG as in Figure 7. however An anti-fgG horseradish peroxidase monoclonal antibody was used. Growth IgG antibodies rose more strongly in children than in humans. However, although it is not noticeable , IgG antibody titers tended to be higher in women with normal elasticity than in women. .

139 serum from a healthy adult woman, C3 serum from a CIN grade 10 woman, C 16 sera from women with IN grade 2, 16 sera from women with CIN grade 3 and 69 sera from women with invasive cell carcinoma of the intestine of the neck to IgA, IgG and ■ gM antibodies were analyzed. CIN and SCC groups for probabilistic analysis 1 were combined into this single cervical tumor group. CIN that matches your age and gender As shown in Figure 9, IgA antibody levels were lower in CIN or Ga compared to the comparison without. It was markedly increased in the tumor patient group. This figure shows someone with CIN or a neck. Figure 2 shows the detection of rgA antibodies in serum from patients with intestinal cancer. blood The serum was diluted 1:2Q and added to the coaching site play at BPV. Ho IgA antibodies were detected with Sladitz peroxidase monoclonal antibody.

Absorption was read at 415 nm 60 minutes after adding the dough. Each point represents the average absorption. vinegar. titels in the CIN and carcinoma group compared to the no CIN group. (p<0.025).

As shown in Figure 10, compared to the comparison, cervical tumor patients showed higher antibody resistance to PVIgG. Tellus was significantly decreased (p<0.

01). This figure shows IgG anti-antibodies in serum from a patient with CIN or cervical carcinoma. Indicates body detection. against BPVI using rabbit anti-human IgG alkaline phosphatase The same sera as in Figure 9 were tested for the presence of gG antibodies. 9 minutes after adding the dough Absorption was later recorded at 405.

According to Figure 11, the PVIgM response in the cervical tumor patient group was higher than that in the comparison group. A significant decrease was observed in the tellus. This figure shows patients with CIN or cervical carcinoma. Detection of anti-PVIgM antibodies in serum from a patient who underwent treatment. Figure 9 shows the same serum Tested for the presence of IgM antibodies. Anti-human IgM glucose oxidizer Plates were read at 415 nm after 60 minutes of incubation using a Nzikogate. Ig Differences between A and IgG responses compared to CIN or cancer layer groups. However, a surprising increase was observed in the CIN or carcinoma groups. Figure 12 is a disease This shows the difference in the above reactions in relation to Same absorption value as in Figure 9.10 was recorded as IgA subtracted by IgG.

All HPV genomes (He1lo es) contain eight protein regions. It has an open reading frame. 2 This 0RFSLl and L2 was shown to encode a protective membrane protein. L1 protein is approximately A 54kDa extra protein protective membrane protein that is effective against both IgG and IgA antibodies. It is immunogenic. Antibodies against the Ll protein are effective at least for BPV-1. It has virus-neutral activity. Various conventional polishing methods using monoclonal etc. Studies have shown that the L1 protein is common to all types of PV and belongs to a so-called group. It has a specific shrimp tobe (apltope), and each HPV type also has an apltope. It has pitobes. Using small fused proteins to identify one group and one Type-specific epitobes were mapped. L20RF contains approximately 70kDa protein. It encodes a protective membrane protein and was termed by us an approximately 64 kDa protein. child This has a relatively low redundancy.

The L2 protein has been reported to have type-specific epitobes. HPV1 This was done to obtain a complete map of the linear epitopes of the six major protective membrane proteins. The amino acid permutations of the two proteins were converted into a 20-amino acid synthesis sequence with 5 amino acid overlaps. It was synthesized as a peptide. IgA from the serum of patients with HPV16 cervical tumors, The positions of permuted epitopes that reacted with IgG or IgM antibodies are shown in Figures 13-19. Shown below.

The deduced amino acid permutations of HPV16 Ll and L20RF result in five amino acids. Sixty-six 20 amino acid proteins with amino acid overlaps were synthesized. protein The putative monitoring hood was designated as amino acid No. 1 to indicate the position within.

Synthetic peptide number 1 corresponds to amino acids 2-21 in L10RF, and peptide number 2 corresponds to amino acids 17-36, number 3 corresponds to amino acids 32-51, L1 Carboxyterminal peptide (number 35) corresponds to amino acids 512-531 .

Peptide number 36 corresponds to L20RF (amino acids 2 to 21) amino terminal do. Number 37 corresponds to amino acids 17-36. L2 Carboxy Terminus ( The two peptides in numbers 65 and 66) were synthesized as 21-residue peptides. .

This makes these amino acid positions 4327-457 and 453-473. Ru. The amino acid permutations of many immunoactive peptides are shown in Table 1. The symbol there is 2 explained in the table.

The amino acid permutations of all peptides used in Figures 13-19 are shown in Figures 13-19. show. Peptides are t-BOC amino acids (Baham AG, Bubendorf, S. ) and p-methylbenzihytriamine resin (Fulkaage, Bach, Switzerland) ) was synthesized by multiple solid-phase peptide synthesis method. formyltribetophane and removal of protecting groups from methionine sulfoxide residues is 25% higher. This was achieved by disruption with drogen fluoride. then peptide is split from the resin using a double tank device with liquid hydrogen fluoride. Ta.

62 from patients with carcinoma (CIN grade 3) or invasive cervical carcinoma serum was obtained. Southern plot of clone HPVI 6 and HPV18 (3 Corresponding cervical bioplasty by dot plot (2 cases) or dot plot (32 cases) (biopsies) were analyzed for the presence of HPV DNA.

HPV16 was detected in 4 out of 32 cases, and HPV18 in 4 cases. hpv1 6. If you collect 30 serum samples from patients with infected neck tumors, all the synthetic peptides will be produced. It was sufficient for testing on chido.

Twenty-two sera were obtained from patients with other tumors. 38 serum healthy women Obtained from.

Synthetic peptides were diluted in 10mM carbonate buffer and 20μg peptide/m 1 to a half-Asian 96-well microfilter plate. P Rates were kept overnight at room temperature. One wash with PPB50.05% Tween After that, plates were blocked with 10% lamb serum in PBS. After that 60 minutes 3 It was left at 7°C. Discard the blocking solution and wrap the plate completely in paper. is. Dilute human serum 1:30 with 10% lamb serum/PBS and incubate at 37°C for 120 minutes. It was allowed to react for a while. The plates were then washed 5 times with PBS-T. Detection of bound antibodies Antibody 1gA horseradish 1peroxy in 10% lamb serum/PBS for Dase monoclonal antibody was diluted 1:500 and plated at 37°C for 120 min. I left it alone. The plate was then washed 5 times with PBS-T and 20 mg/m 1 of 2.2'-azinodi(3-ethylbenzthiazoli n-sulfonat( 6)) Cultured with diammonium salts (0° l in 1M citrate buffer; 50 dilution) .

Absorption was recorded after 60 minutes at 415 nm. Wash the plate for IgG detection. The cells were washed and blocked with 10% lamb serum-PBS at 37°C for 60 minutes. Then rabbit anti-human body GG alkaline conjugate (1000 diluted in 10% lamb serum-PBS) ) was applied for 120 minutes at 37°C. 5 times with PBS-T and 0.1M jetanolamine After one wash with buffer, 1 mg/g in 0.1 M diethanolamine buffer. m1 phosphatase dough was added and the plate was read at 404 nm after 90 minutes. .

For detection of IgM antibodies, plates were washed and blocked as above, then 0% Lamb Serum - PBSI: Antibody 1gM Glucose Oxidase in 80G Dilution It was left for 120 minutes at Njiko Gate. 0.36mg/ml ABTS as dough 2.4% glucose, 8μg/ml in 0.1M phosphate buffer Radishpa oxidase was used. Read the plate after 60 minutes at 415 nm. is. Absorption of 0.1 or more was considered a positive reaction. As an internal standard, 16 positive blood The antigen peptide HKSsl from E20RF of HPVI 6 was detected in each test. 0ELISA absorption that reacted with VTLYYDSEIQRDQC was adjusted to internal standards. were prepared to compensate for analyte-to-analyte variation.

The probabilistic relationship between the patient group with HPV-infected lesions and the comparison group There are significant differences in EL I SA absorption compared to uncoated well fabrics. The absorption of the same serum was also analyzed.

HPV type 16 as a series of 66 synthetic peptides, 20 amino acids long and 5 residues overlapping The amino acid permutations of the Ll and L2 proteins were synthesized. All peptides are HPV1 6 Regarding reactivity with Igk or IgG antibodies in 30 sera from infected cervical tumor patients. and tested by ELISA. As shown in Figure 13, how many L1 proteins This region showed reactivity. This figure shows Igk reactivity to L1 protein. . Each point is the absorption value for this serum that reacted with the peptide numbered on the horizontal axis. shows. Subtract the absorbance value of the same serum when reacting with uncoated wells. there was. ) (30 sera from patients with PV16-infected neck tumors were diluted 1:30). 35 20 residues representing all amino acid permutations of LIORF of HPVI6. The base was reacted with a synthetic peptide. IgA specific specific engine complex lonal antibody-binding a antibody was detected.

Inside protein expanded amino acids 167-271 (corresponding to peptides 12-18) Igk reactions were particularly strong for peptides from this region. This main immune activity Outside the region there were several additional immunoreactive epitopes. Especially tan, f amino terminal (peptide l), peptide 8 (amino acids 102-121) and main Some shrimp and peptides located at the carboxyl terminal of the immunoactive region Do 21 and 3! (corresponding to amino acids 302-321.452-471) etc. Admitted.

In comparison, the peptides derived from the L2 protein in the serum shown in Figure 14 There was almost no reaction with the IgA antibody inside. This figure shows I for L2 protein. Shows gk reactivity.

Thirty-one 20-residue synthetic peptides representing all amino acid permutations of HPV L20RF. An experiment similar to that in Figure 13 was conducted, except that the titanate and serum reacted.

The main epitope is in the middle of the protein (peptide 49, amino acids 197-216) It was located in Other shrimp topes are tannokuri (peptides 37-38, amino acids 17 to 41) were detected to be located at the amino terminal portion. L2 carbo The xyl terminal was less reactive. IgG reactivity of L1 tannokuri ! t Lower than Igk reactivity. Figure 15 shows the IgG reaction against one L1 tank. shows responsiveness. The experiment was conducted in the same way as the experiment in Figure 13, except that IgG (gamma chain) ) except that a specific enzyme-conjugated rabbit antibody was used. Main Ig A immunoactive region (peptide 12-1g) is immunoactive with IgG antibodies, and IgG Reactivity is observed outside this region (e.g. peptide 24 (amino acids 347-366)). It was the same as Ebitobe.

As shown in Figure 16, the L2 protein has a slight IgG-reactive epitope. Ta. This figure shows IgG reactivity to L2. IgG (gamma chain) specific enzyme Using a seam-conjugated rabbit antibody, serum was tested for all HPV L20R amino acids. Apart from reacting with F31 20-residue synthetic peptides representing permutations, The same experiment as in Figure 13 was conducted. This result indicates the Igk reactivity of this protein. It was similar to what we discovered. This protein is mainly Igk-reactive. It was confirmed that a certain peptide 49 is an IgG-reactive epitobe. L IgA reactivity at the amino terminus of 2 (peptides 37-38) IgG reactivity was confirmed (Fig. 4).

As shown in Figure 17, IgM reactivity towards L1 protein increases along the entire length of the protein. It was distributed over several Ebitobe. This figure shows the I for L1 protein. Indicates gM reactivity.

Except that an IgM (mu chain) specific enzyme-mucodium gate goat antibody was used. Then, an experiment similar to that shown in FIG. 13 was conducted. Interestingly, IgG or I The main 1 gM reactivity is for peptides that are less immunoreactive for gA antibodies. confirmed.

1gM immunoreactivity of L2 protein indicates that L2 peptide is less reactive than L1 peptide. It was similar for IgA and IgG. Figure 18 shows 1g for L2 protein. Shows M reactivity.

1 gM (mu chain) specific enzyme mucophagi 1 gate goat antibody was used to detect blood clots. Thirty-one 20 residues represent all amino acid permutations of HPV16 L20RF. An experiment similar to that in FIG. 13 was conducted except that the reaction was with a synthetic peptide.

One L2 peptide among 4 or more from 30 sera is not IgM and has no immunoreactivity. There wasn't. The seven most immunoactive peptides (8, 12, 13, 14, 16, 17 and 24) using a panel of 60 comparative sera for gA, IgG and 1gM reactivity. I tested it. Of these, 22 were from patients with unrelated tumors and 38 were from healthy patients. Obtained from subject. Most of these patients showed significant immunoreactivity. comparison blood As for pure water, it was less than 10%. Figure 19 shows the disease reactivity of synthetic peptides. vinegar.

As shown in Figure 19, four of the highly immunoactive synthetic peptides are 30HPV18. from patients with tainted cervical tumor serum (HP'V16SCC) and other intestinal tumors A comparison group of 60 sera from healthy subjects showed a highly reactive showed the difference. Each point is the EL I after subtracting the absorption value of the non-footed well. Indicates the absorption value in SA. With 1gM reactivity to peptide 8nia 0%, High sensitivity was observed for detecting HPV16-infected cervical tumors. This test Speefficity is 95% (3/60 comparative serum reacts) ). 1 gM reactivity towards peptide 24 provides a high degree of specificity. showed = 97% (2/60 comparison sera were reactive). However, the sensitivity was low =53% (16/30 patient sera reacted). Ig against peptide 14.16 The sensitivity of A reactivity was 60% and the specificity was slightly above 90% (Kan 1 Figure 9). Immunoreactivity in positive sera was among the highest detected (No. (Compare with figures 1 to 6).

By demonstrating that the peptide is immunoactive, we have identified an epitope that reacts with human serum. There are some other examples, including one. The epitobe contained in this peptide permutation is Although not absolutely dependent on the exact permutation of the peptides, slight variations in the original peptide may It is possible for a long tooth to be lost in a person who is in a good condition. Such metamorphosis includes elongation, contraction and contraction. , cyclization and amino acid substitution. Peptides sometimes so transformed There is a suspicion that this may be a new peptide containing a new Ebitope. I have a question. Comparative immunoassays between the original peptide and the transformed peptide revealed that the transformed peptide If the initial peptide is substantially immunoreactive against antibodies and therefore the same epitope Determining whether or not it contains Tove is easy. Peptides can be used in a variety of ways. The point that it can be manufactured should be emphasized. Peptide synthesis using organic chemical methods was adopted. The same peptides have been used in other ways, such as recombinant DNA expression systems. It can also be manufactured by

This invention is not limited to the above description. Immunological analysis can be performed using various methods, e.g. It can be done with For example, detection of antibodies bound to a specific antigen can be done using various antibodies. Has affinity for antibodies such as counter antibodies (anti-antibodies) and peptide A and peptide B. This can also be achieved using other compounds. Labeling of these reactants (1abel 1 ing), such as fluorescein (fluoroimmunoassay) and encima ( Enzyme-linked immunospecificity, ELISA or EIA) to radioactivity It can also be done using methods such as (radio immunoassay). Special Enseem Immunity For analysis, antigen-antibody complexes may be detected on muscle. hands like this The method is called immunostaining or immunohistochemistry, but its principle is based on ELIS. Same as A.

ELISA methods can be performed in a variety of formats.

Multiple layers of anti-antibody, avidin-biotin conjugate and enzyme anti-ensieme conjugate The method used is known. The rigid support that immobilizes the antibody is usually plastic. Ru. However, other materials such as latex and agarose are also used. You can also use antibodies directly. It does not need to be fixed to a rigid support. For example, catching or sandwich E A solid-phase immobilized antigen antibody called LISA may also be used.

Immunoassay that plots two antigens on a sheet-like rigid support is called immunoblotting. It is. Typically this rigid support is nitrocellulose or nylon sheet. However, other supports may be used. In this case, the antigen should be added before blotching. Separate them using gel electrophoresis according to their size. certain on the sheet Detection of antibody binding to antigen is performed in the same way as in immunoassays.

Diagnostic methods are generally difficult (sensitivity and specificity can be improved by combining some methods). Therefore, the various methods described above can be combined as appropriate. It is possible to match.

Table 1A IgG serum secretion 19084, 368, 184 190g5, 592, 173 190B6 1.041, 306 19087. 230. 0)3 190g! 1. 770. 111g 19089, 240, 180 19090, 253, 294 19091. 210. 032 19092. 198. 060 19093, 660, 195 19G94. 945. 2a1 19095, 224, 228 19096. 280. 278 19097, 259, 226 1909g, 222. 134 IgA serum secretion 19084, 298, 090 19085, 199, 072 1908B, 327, 131 190g? , 137. 056 19088, 235, 118 190g9, 140. 053 19090, 153, 070 19091, 12! , (176 19092, 119, 052 190931,021,394 19094, 140, 068 19095, 073, 099 19096,136,098 19097, 128, 096 190911, 098, 050 Measurement of IgG antibodies against PVIgAS in serum and secretions.

The extinction factor from EL I SA is based on 15 patients. Some of this information ．． Shown in Figure 3.4. Probabilistic analysis of the data shows that in PVIgA versus anti-IgA serum There is no correlation with the results of the anti-PVI ggG antibody test, whether it is present in the body or in secretions. death.

Table 1 Pep peptide IgA IgG IgM tied permutation 8 NKFGFPDTSFYNPDTQRLVW <0.05 <0.0001 <0.000112 VDNRECISMDII[QTQLCLIG><0. 002 <0.0001 <0.0113 LCLIGCKPPIGEHIGK OSPC<0.02<0.0001 N514 KGSPCTNVAVNPG DCPPLEL < 0.0005 < 0.0001 < 0.000116 VBTGFGAMDFTTLQANKSEV <0.0001 <0.000 1 <0.000117 NKSEVPLDICTSICKYPDY+ <0. 01 NS <0.000124 NGICWGNQLFVmDTTRST < 30 patients with cervical requirements of 0.002 <0.00Q1 <0.00116 The significantly increased anti-HPV16 synthetic peptide antibody titers in serum from patients with other tumors Comparison with 60 sera from patients with tumors etc. The figure shows the manifestation of these two parameters. NS showing a significant difference in Xp value = not significant Table 2 Symbol Amino acid F Phe L phenylalanine M Met L methionine A Ala L Alamin S Ser L Serine 1 11e L isoleucine L Leu L Leucine T Thr L threonine V Val L Valine P Pro L Proline K Lys L lysine HHis L Histidine Q Gln L glutamine E Glu L glutamic acid W T r y L) Ribtofan RArg L-arginine D Asp L aspartic acid N Asn L Asparagine CCys L-cystine Table 3 Number permutation I QVTFIYILVITCYE[)VNVY2 DVNVYHIFFQMS LWLPSEA Ding 3 PSEA Ding VYLPPVPVSKVVSTD4 VVST DEYVARTNIYYHAGTS5 HAGTSRLAVGHPYFPIK rP6 PrKKPNWNmVPKVSGLQY7 5GLQYRVPRIHL PI) PNKFGF8 NKFGFPDTSFY? 1PDTQRLVW9 QR I, VIFACVGVEVGRGQPLGVlo QPLGVGISGBPLL NKLDDTell LDDTENASAYAANAGVDNRE12 VDN RECISMDYKOTOLCLIG13 LCLIGCKPPPTGEHWGK GSPC141[GSPCTNVAVNPGDCPPLEL15 PPLELI NTVIQDGDMVHTGF16 VHTGFGAMDPTTLQAN! l5 EV17 NKSEVPLDICTSI (JYTDY118 YPDYI [MV SEPYGDSLFFYL19 LFFYLRREQMPVRIILFNRAG 20 FNRAGTVGENVPDDLYIKOS21 YIKGSGSTAN LASSNYFPTP22 YFPTPSGSMVTSDAQIFNKP23 rFN■PYWLQRAQGHNNGICF24 NGICIGNQI, FVT VVDTTR3T25 TTR5TNMSLCAAIS 5ETTY25 5E TTYINTNFXEYLRHGEEY27 HGEEYDLQFIFQLCK ITLTA2g 1TLTADVMTYIHSMNSTILE29 STILE DWNEGLQPPPGGTLRE30 GGTLEDTYRFVTQAIAC QKIII31 ACQKHTPPAPKEDDPL■YT32 LIHYTF WEVNIJEKFSADLD33 5ADLDQFPLGRKFLLQAGI J34 QAGIJAKPKFTLGKRKATPT35 KATPTTSST STTAKRKKRKL36 RHKRSAKRTKRASATOLYKT37 QLYITCKQAGTCPPDIIPKV3g +1PIVEGKTIAE QILQYGSM39 QYGSMGVFFGGGLGIGTGSGT40 TG SGTGGRTGYIPLGTRPPT41 TRPPTATDTLAPVRP PLTVD42 PLTVDPVGPSDPSIV SLVEE43 5LVEE TSPIDAGAPTSVPSI44 5VPSIPPDVSGFSITTST DT45 TSTDTTPAILDINNYVYYVT46 VTTVTTII INNPTFTDPSVLQP47 5VLQPPTPAETGGHFTLSS S48 TLSSSTISTHNYEEIPMD-F49 PMDTFIVST NPNTVTSSTPISo 5STP+PGSRPVARLGLYSRT51 LYSRTTQQVKVVDPAFVTTP52 FVTTPTKLITYD NPAYEGID53 YEGIDVDNTLYFSSNDNNSIN54 DN SINIAPDPDFLDIVALHR55VALIIRPALTSRRTGI RYSRI56 RYSRIIGNKQTLRTRSGKSIG57 GKSIG AKV[(YYYDLSTIDPA58 TIDPAEEIELQTLTPST YTT59 5TYTTTSHAASPTSINNGLY60 NNGLYDI YADDFITDTSTTP61 TSTTPvPSVPSTSLSGYIPA 62　GYIPANTT[PFGGAYNIPLV63　NIPLVSGPDI PINITDQAPS64 DQAPSLIPIVPGSPCYTIIA65 YTIIADAGDFYLHPSYYMLRK66 YMLRKRRKRLPY FFSIVSLAA

Claims (40)

[Claims] 1. Approximately 64 kDa papilloma virus protein code L2 and approximately 54 kDa papilloma virus protein code L2 Antibodies against virus protein code L1 and approximately 28 kDa papilloma virus protein Antibodies against various codes and antibodies against the approximately 14 kDa papilloma virus protein. Taha [There is an array] a peptide having an amino acid permutation represented by a formula selected from the group consisting of; is a modification of a peptide from one of these groups and an antibody against the initial peptide Confirm the presence of IgG antibodies in body fluids or muscles that are immunoreactive with Perform detection by Human papilloma virus (HPV) for medical purposes characterized by Method for preventing tumors with PV on muscles. 2. Antibodies originate from the human body and the infection to be detected involves HPV infection or HPV is sick The method according to claim 1, characterized in that: 3. 2. The method according to claim 1, wherein the presence of antibodies is determined by immunoassay. Method. 4. Immunoassay is ELISA 4. A method according to claim 3, characterized in that. 5. The immunoassay is characterized by immunoblotting. The method according to claim 3. 6. Detection is carried out by secretions The method according to claim 1, characterized in that: 7. The method according to claim 6, characterized in that the detection is performed on cervical secretions. Law. 8. The method according to claim 7, wherein the detection is performed by diagnosing a cervical tumor. Law. 9. Secretions are collected with sterile cotton, a brush, a spatula, etc., and then washed with a diluted solution. Ru 7. The method according to claim 6, characterized in that: 10. Detection is done on serum The method according to claim 1, characterized in that: 11. Approximately 28 kDa papilloma virus protein and approximately 14 kDa papilloma virus protein Antibodies against proteins or [sequences available] a peptide having an amino acid permutation represented by a formula selected from the group consisting of; is a modification of a peptide from one of these groups and an antibody against the initial peptide Confirm the presence of IgG antibodies in body fluids or muscles that are immunoreactive with Perform detection by The method according to claim 1, characterized in that: 12. The antibody originates from the human body and the infection to be detected is an HPV infection or involves HPV. I have a disease 12. The method according to claim 11. 13. 12. The method according to claim 11, wherein the presence of antibodies is determined by immunoassay. How to put it on. 14. Immunoanalysis is ELISA 12. The method according to claim 11. 15. The immunoanalysis is characterized by immunoblotting. 12. The method according to claim 11. 16. Detection is carried out by secretions 12. The method according to claim 11. 17. 17. Claim 16, characterized in that the detection is carried out on cervical secretions. the method of. 18. Claim 17, wherein the detection is performed by diagnosing a cervical tumor. the method of. 19. Secretions are collected with sterile cotton, a brush, a spatula, etc., and then washed with a diluted solution. be able to 17. The method according to claim 16, characterized in that: 20. Detection is done on serum 12. The method according to claim 11, characterized in that: 21. [There is an array] a peptide having an amino acid permutation represented by a formula selected from the group consisting of; is a modification of a peptide from one of these groups and an antibody against the initial peptide Confirm the presence of IgG antibodies in body fluids or muscles that are immunoreactive with Perform detection by The method according to claim 1, characterized in that: 22. The antibody originates from the human body and the infection to be detected is an HPV infection or involves HPV. I have a disease 22. A method according to claim 21, characterized in that. 23. 22. The method according to claim 21, wherein the presence of antibodies is determined by immunoassay. How to put it on. 24. Immunoassay is ELISA 24. A method according to claim 23, characterized in that. 25. The immunoanalysis is characterized by immunoblotting. 24. The method according to claim 23. 26. Detection is carried out by secretions 22. A method according to claim 21, characterized in that. 27. Claim 26, characterized in that the detection is carried out on cervical secretions. the method of. 28. Claim 27, wherein the detection is performed by diagnosing a cervical tumor. the method of. 29. Secretions are collected with sterile cotton, a brush, a spatula, etc., and then washed with a diluted solution. be able to 27. A method according to claim 26, characterized in that. 30. Detection is done on serum 22. A method according to claim 21, characterized in that. 31. Approximately 28 kDa papilloma virus protein and approximately 14 kDa papilloma virus protein Antibodies against white or [there is a sequence] a peptide having an amino acid permutation represented by a formula selected from the group consisting of; is a modification of a peptide from one of these groups and an antibody against the initial peptide Confirm the presence of IgG antibodies in body fluids or muscles that are immunoreactive with Perform detection by The method according to claim 1, characterized in that: 32. The antibody originates from the human body and the infection to be detected is an HPV infection or involves HPV. I have a disease 32. A method according to claim 31, characterized in that. 33. 32. The method according to claim 31, wherein the presence of antibodies is determined by immunoassay. How to put it on. 34. Immunoassay is ELISA 34. A method according to claim 33, characterized in that. 35. The immunoassay is characterized by immunoblotting. 34. The method of claim 33. 36. Detection is carried out by secretions 32. A method according to claim 31, characterized in that. 37. 37. Claim 36, characterized in that the detection is carried out on cervical secretions. the method of. 38. Claim 37, wherein the detection is performed by diagnosing a cervical tumor. the method of. 39. Secretions are collected with sterile cotton, a brush, a spatula, etc., and then washed with a diluted solution. be able to 37. A method according to claim 36, characterized in that. 40. Detection is done on serum 32. A method according to claim 31, characterized in that.